PROJECT SUMMARY Epidemic typhus, also known as Gaol fever, Jail fever, or Famine fever, is caused by the Gram-negative bacterium Rickettsia prowazekii. This louse-borne disease has been responsible for devastating widespread epidemics throughout history, especially during and after the wars, famine, and poor socio-economic conditions, and is the only known rickettsiosis that can recur after a long period of latency (Brill-Zinsser disease). R. prowazekii is classified as a CDC/NIAID Category B biological warfare pathogen, the entire genome for which was the first to be sequenced among all known Rickettsia species. Despite considerable restrictions imposed by obligate intracellular parasitism, tremendous strides have recently been made in the genetic manipulation of R. prowazekii. Strikingly, the knowledge of molecular aspects of interactions with the vascular endothelium, the preferred cell type infected during human infections, still remains in its infancy. It is noteworthy that endothelial cells, which are intimately involved in the manifestations of rickettsial infections, have emerged as key immunoreactive cells involved in host defense and inflammation. On the basis of interactions of host endothelial cells with R. prowazekii and R. typhi (the etiologic agent of endemic typhus), we have identified activation of nuclear factor kappa B (NF-[unreadable]B) and stress-activated p38 protein kinase as critically important regulatory signaling mechanisms that contribute to host cell activation and responses to infection. The first aim of this exploratory grant application is designed to define the extent and kinetics of the activation of NF-[unreadable]B and MAP kinase signaling pathways after R. prowazekii infection of vascular endothelial cells in vitro and to test the hypothesis that potential differences in intracellular signaling mechanisms determine the intensity of host cell activation in response to virulent Breinl versus attenuated Madrid E strains of R. prowazekii. Aim 2 will focus on establishing and characterizing a mouse model of R. prowazekii infection that closely mimics the major pathological features, i.e. disseminated endothelial infection and vascular inflammation, of epidemic typhus disease in humans. The long-term objective of this project is to lay the foundation for comprehensive understanding of epidemic typhus pathogenesis by identifying specialized vascular cell signaling pathways activated in vitro and subsequent detailed analysis of their involvement in determination of innate and adaptive immune responses with an aim to develop unique chemotherapeutic strategies focused at targeted intervention. In addition, detailed characterization of a small animal model of infection akin to disease in humans will allow us to define unique features of epidemic typhus pathogenesis and virulence factors of R. prowazekii, to expand our understanding of in vivo host immune responses, and to test the efficacy of novel antibiotics and vaccine candidates.